T he concentration of salt in the thin layer of fluid at the surface of large airways, the airway-surface liquid (ASL), is believed to be of central importance in airway physiology and in the pathophysiology of cystic fibrosis. Invasive sampling methods have yielded a wide range of ASL [NaCl] from 40 to 180 mM. We have developed novel fluorescent probes and microscopy methods to measure ASL thickness, salt concentration, and pH quantitatively in cell-culture models and in the trachea in vivo. By rapid z-scanning confocal microscopy, ASL thickness was 21 ± 4 μm in well-differentiated cultures of bovine tracheal epithelial cells grown on porous supports at an air-liquid interface. By ratio imaging fluorescence microscopy using sodium, chloride, and pH-sensitive fluorescent indicators, ASL [Na⁺] was 97 ± 5 mM, [Cl^–] was 118 ± 3 mM, and pH was 6.94 ± 0.03. In anesthetized mice in which a transparent window was created in the trachea, ASL thickness was 45 ± 5 μm, [Na⁺] was 115 ± 4 mM, [Cl^–] was 140 ± 5 mM, and pH was 6.95 ± 0.05. Similar ASL tonicity and pH were found in cystic fibrosis (CFTR-null) mice. In freshly harvested human bronchi, ASL thickness was 55 ± 5 μm, [Na⁺] was 103 ± 3 mM, [Cl^–] was 92 ± 4 mM, and pH was 6.78 ± 0.2. These results establish by a noninvasive approach the key properties of the ASL and provide direct evidence that the ASL is approximately isotonic and not saltier in cystic fibrosis.